Hi Sam,
The effect of reducing agents like beta-ME, DTT or TCEP is exactly as you say,
the prevention of disulphide formation or cleavage of existing disulphide
bridges and therefore prevention of aggregation if inter-molecular disulphide
bridges can be formed.
A balancing of reducing power, or better, the redox potential will be
necessary
if you want to reduce certain cystein residues while leaving others
oxidized as
dimers. For example, you have a disulphide containing, secreted protein fused
to a TEV-cleavable tag. The Cystein protease will have to be kept reduced,
while the disulphide bridges of the target protein must be spared. For cases
like that I found it useful to use a mixture of reduced and oxidized
glutathione to adjust the redox potential ('redox buffer').
If the reducing agent is necessary to keep the protein happy, I would not
dialyse it away, at least not for the first screening experiments. For beta-ME
one should keep in mind that it has a certain potential to form adducts with
the proteins cystein residues.
Cheers,
Clemens
Quoting Sam <[EMAIL PROTECTED]>:
Dear all,
Can anyone enlighten me the effect of reducer in crystallography?
I understand that it removes disulphide bond, and prevent protein
aggregation. But how do we find a balance point and must we remove it before
screening crystals?
Thanks for answering first.
Cheers
sam
